The production of intracellular polyesters belonging to the class of polymers known as polyhydroxyalkanoates (polyhydroxyalkanoic acids) has been observed in a wide array of prokaryotic organisms (Anderson, A. J. and Dawes, E. A. (1990) Microbiol. Rev. 54:450-472; Steinbüchel, A. and Valentin, H. E. (1995) FEMS Microbiol. Lett. 128:219-228). The monomers composing the polyesters range in length from C4 (3-hydroxybutyrate) to C12 (3-hydroxydodecanoate) (Lageveen, R. G. et al. (1988) Appl. Env. Microbiol. 54:2924-2932). These polyesters have attracted considerable interest as they are biodegradable. Potential technical applications exist in industry and agriculture, as well as in medical devices and procedures (Hocking, P. J. and Marchessault, R. H. (1994) Biopolyesters. In: G. J. L. Griffin (Ed) Chemistry and technology of biodegradable polymers, Chapman & Hall, London, pp.48-96; Müller, H. M. and Seebach, D. (1993) Angew. Chem. 105:483-509). Additionally, this class of polyesters is attractive as a potential alternative to conventional petrochemical-derived plastics.
Polyhydroxyalkanoic acids are broadly characterized according to the monomers that constitute their backbone. Polymers composed of C4-C5 units are classified as short chain length (scl) polyhydroxyalkanoic acids; polymers containing monomers of C6 units and above are classified as medium chain length (mcl) polyhydroxyalkanoic acids. The primary structure of the polymer influences the physical properties of the polyester.
The metabolic pathways leading to the formation of polyhydroxyalkanoic acids have not been elucidated for all organisms. The most extensively studied polyhydroxyalkanoic acid biosynthetic pathway is that of Alcaligenes (Peoples, O. P. et al. (1989) J. Biol. Chem. 264:15298-15303; Valentin, H. E. et al. (1995) Eur. J. Biochem. 227:43-60). This organism is capable of forming either a homopolymer of C4 (polyhydroxybutyrate, PHB) or a co-polymer of C4-C5 (PHB-PHV, polyhydroxybutyrate-polyhydroxyvalerate) (Koyama, N. and Doi, Y. (1995) Biotechnol. Lett. 17:281-284). Hence, A. eutrophus is classified as a scl polyhydroxyalkanoic acid organism. Similarly, Pseudomonas species generate a polymer composed of monomers ranging in length from C6 to C12 (Timm, A. and Steinbüchel, A. (1990) Appl. Environ. Microbiol. 56:3360; Lageveen, R. G. et al. (1988) Appl. Env. Microbiol. 54:2924-2932), and are classified as mcl polyhydroxyalkanoic acid organisms.
The polymerization of the hydroxyacyl-CoA substrates is carried out by polyhydroxyalkanoic acid synthases. The substrate specificity of this class of enzyme varies across the spectrum of polyhydroxyalkanoic acid producing organisms. This variation in substrate specificity of polyhydroxyalkanoic acid synthases is supported by indirect evidence observed in heterologous expression studies (Lee, E. Y. et al. (1995) Appl. Microbiol. Biotechnol. 42:901-909; Timm, A. et al. (1990) Appl. Microbiol. Biotechnol. 33:296-301). Hence, the structure of the backbone of the polymer is strongly influenced by the polyhydroxyalkanoic acid synthase responsible for its formation.
Fluorescent pseudomonads belonging to the rRNA homology group I can synthesize and accumulate large amounts of polyhydroxyalkanoic acids (PHA) composed of various saturated and unsaturated hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (Steinbüchel, A. and Valentin, H. E. (1992) FEMS Microbiol. Rev. 103:217). Polyhydroxyalkanoic acid isolated from these bacteria also contains constituents with functional groups such as branched, halogenated, aromatic or nitrile side-chains (Steinbüchel and Valentin (1995 FEMS Microbiol. Lett. 128:219-228). The composition of polyhydroxyalkanoic acid depends on the polyhydroxyalkanoic acid polymerase system, the carbon source, and the metabolic routes (Anderson, A. J. and Dawes, E. A. (1990) Microbiol. Rev. 54:450-472; Eggink et al. (1992) FEMS Microbiol. Rev. 105:759; Huisman, A. M. et al. (1989) Appl. Microbiol. Biotechnol. 55:1949-1954; Lenz, O. et al. (1992) J. Bacteriol. 176:4385-4393; Steinbüchel, A. and Valentin, H. E. (1995) FEMS Microbiol. Lett. 128:219-228). In P. putida, at least three different metabolic routes occur for the synthesis of3-hydroxyacyl CoA thioesters, which are the substrates of the polyhydroxyalkanoic acid synthase (Huijberts, G. N. M. et al. (1994) J. Bacteriol. 176:1661 -1666): (i) β-oxidation is the main pathway when fatty acids are used as carbon source; (ii) De novo fatty acid biosynthesis is the main route during growth on carbon sources which are metabolized to acetyl-CoA, like gluconate, acetate or ethanol; and (iii) Chain elongation reaction, in which acyl-CoA is condensed with acetyl-CoA to the two carbon chain extended β-keto product which is then reduced to 3-hydroxyacyl-CoA. This latter pathway is involved in polyhydroxyalkanoic acid-synthesis during growth on hexanoate.
The polyhydroxyalkanoic acid synthase structural gene from Alcaligenes eutrophus (phaCAe) has been cloned and characterized at the molecular level in several laboratories (for a review see Steinbüchel, A. and Schlegel, H. G. (1991) Mol. Microbiol. 5:535-542; GenBank Accession number J05003). It was demonstrated that phaCAe in combination with other genes conferred the ability to synthesize poly(3-hydroxybutyric acid) not only to many bacteria, which do not synthesize this polyester such as e.g. Escherichia coli (Steinbüchel, A. and Schlegel, H. G. (1991) Mol. Microbiol. 5:535-542) but also to Saccharomyces cerevisiae (Leaf, T. A. et al. (1996) Microbiology 142:1169-1180), plants such as Arabidopsis thaliana (Poirier, Y. et al. (1992) Science 256:520-523.) and Gossypium hirsutum (John, M. E. and Keller, G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:12768-12773), and even to cells from the insect Spodoptera frugiperda (Williams, M. D. et al. (1996) Appl. Environ. Microbiol. 62:2540-2546).
The development of biological systems that synthesize poly (4-hydroxybutyric acid), poly (3-hydroxybutyric acid-co-4-hydroxybutyric acid), and other polyester materials would be of great utility. Biological systems provide the potential to produce significant quantities of important materials, while utilizing inexpensive feedstocks and minimizing hazardous byproducts.
There exists a need for novel biosynthetic routes to polymers of potential commercial interest that do not rely on petroleum based starting materials. Biological processes present an attractive alternative to chemical processes that produce potentially harmful byproducts while consuming non-renewable resources.